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United States Department of Agriculture

Agricultural Research Service

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Research Project: GENETIC AND BIOLOGICAL DETERMINANTS OF AVIAN TUMOR VIRUS SUSCEPTIBILITY

Location: Avian Disease and Oncology Laboratory

2010 Annual Report


1a.Objectives (from AD-416)
Identify genetic predictors of Marek's disease virus (MDV) virulence. Identify host-viral genetic determinants that control avian tumor virus pathgenicity and shedding. Elucidate the genetic determinants that modulate MDV interactions within the avian immune system. Elucidate host-viral interactions that drive the evolution of new virulent strains of avian tumor viruses. Discover safe and highly effective vaccine platforms that convey protection against emerging MDV strains.


1b.Approach (from AD-416)
Avian tumor viruses of economic importance include:.
1)Marek’s disease virus (MDV), a herpesvirus that induces a lymphoproliferative disease of chickens that, in the absence of effective control measures, is capable of causing devastating losses in commercial layer and broiler flocks; and.
2)avian retroviruses, namely avian leukosis virus (ALV) and reticuloendotheliosis virus (REV); both are associated with neoplastic diseases and other production problems in poultry. Also, both ALV and REV are potential contaminants of live-virus vaccines of poultry. Critical needs are:.
1)better MDV vaccines to protect against the current and next generation of virulent field strains of MDV; and.
2)a long-term strategy designed to reduce the ongoing emergence of new virulent MDV, and creation of recombinant ALVs through multiple barriers or reduction in viral load and shedding. The primary emphasis will be on molecular approaches to better understand which viral genes are important for immunopathogenesis and shedding of MDV. Parallel studies will monitor the virulence of field strains of MDV and ALV. Studies are also aimed at characterization of new virus isolates and on improving assays for their detection; additional efforts will be devoted to better understand MDV immunity. The project also emphasizes studies on:.
1)elucidating factors involved in creation of recombinant ALVs; and.
2)determining whether REV genome insertion into MDV and fowlpox virus influences transmission and epidemiology of REV. The end product will be a better understanding of viral gene function, virus-host interactions and the development of materials and improved methodology for control of avian tumor viruses.


3.Progress Report
Substantial progress was made on all objectives of the project. Brief description of selected accomplishments is listed below. We have used a molecular technique named bacterial artificial chromosome (BAC) to delete a Marek’s disease (MD) virus (MDV) gene, UL39 that encodes for the large subunit of an MDV enzyme known as ribonucleotide reductase (RR). We found that RR is highly expressed in all of MDVs inoculated cell cultures and chickens, regardless of the serotype of MDV. The information should help in our attempts to study MDV gene function and development of new generation vaccines. We have previously reported on insertion of part of genetic material from reticuloendotheliosis virus (REV) named long terminal repeat (LTR) into strain Md5 of MDV using BAC. This year, we examined whether the REV LTR was retained by the rMd5 BAC virus following infection of cell cultures, and one-day susceptible chickens. Results suggest that REV LTR was stable in cell culture, but not in chickens. The loss REV LTR following replication of MDV in chickens appears to occur within the first three weeks, suggesting that the influence of REV LTR on pathogenicity of MDV may not be dependent on retention of LTR. The immunological basis for resistance or susceptibility to MD was examined in highly susceptible and resistant chickens. Analysis of tissue samples collected at different time points post inoculation with MDV revealed a major difference between the two chicken lines in population of immune cells known as CD4+ T; there were more CD4+ T cells in susceptible chickens. This information is essential in understanding of the immunological basis for resistance and susceptibility to MD. Our studies also revealed an MDV oncogene (a gene associated with cancer inducing ability of MDV) named meq can induce down-regulation of many components of the immune system known as cytokines and chemokines that are essential for orchestrating an effective anti-viral immune response. We used restrictive and permissive chicken embryo fibroblasts (CEFs), cells that are resistant and susceptible, respectively to certain subgroups of avian leukosis virus (ALVs) to screen for recombinant ALV (viruses that contain genetic materials from more than one subgroup of ALV). Potential recombination between two subgroups of ALV, namely ALV-J and ALV-A was examined by a DNA-based technique named polymerase chain reaction (PCR). Data suggest that use of restrictive CEFs is an essential step in any scheme for screening for recombinant ALVs. The data also suggest that simple co-infection of CEFs with more than one subgroup of ALV may not necessarily lead to recombinant ALVs and that other factors that may facilitate recombination between two subgroups of ALV should be explored. Finally, we report on our attempts of using MDV-meq-deleted BAC to develop a polyvalent vaccine against MD and infectious laryngotracheitis (ILT), an economically important herpesvirus (ILTV)-induced respiratory disease of chickens. Results indicate that both the gB and gJ genes, genes related to immunity against ILTV were stable, as evidenced by measurable levels of expression of ILTV immunologic proteins.


4.Accomplishments
1. Generation of a recombinant Marek's disease virus (MDV) lacking a protein (enzyme) essential for DNA synthesis. We used a molecular technique known as bacterial artificial chromosome (BAC) to generate a deletion mutant lacking a gene termed UL39 that encodes the large subunit of an enzyme known as ribonucleotide reductase (RR). The active enzyme consisted of both large subunit (RR1) and small subunit (RR2). We developed a specific reagent known as monoclonal antibody T81 for detection of RR expression in MDV infected cells. Using T81 monoclonal antibody, we found RR is highly expressed in all of MDVs inoculated cell culture and chickens, regardless of the serotypes of MDV. This year, we generated a recombinant MDV mutant with a deletion in RR1. Characterization of the RR1 deletion mutant should elucidate its biological function in MD pathogenesis.

2. Currently, Marek’s disease is controlled through the use of vaccines, the majority of which are generated by blindly culturing the virulent virus until it mutates and no longer causes disease. Understanding this process is of both scientific and commercial interest. This year we have generated an attenuated Marek’s disease viruses from a molecular clone. These materials form the basis for identifying the molecular changes that occur in the viral genome that lead to attenuation. If successful, the resulting information will lead to improved vaccines that the poultry industry could use.

3. Further studies of a molecular clone known as bacterial artificial chromosome (BAC) of Marek’s disease virus (MDV) containing part of genetic material termed long terminal repeat (LTR) from an unrelated avian tumor virus named reticuloendotheliosis virus (REV). Recently, we have reported on the development and pathogenicity of a BAC clone of MDV termed rMd5 BAC containing an insert of LTR of REV. In the current study, we examined whether the REV LTR was retained by rMd5 BAC following infection of duck embryo fibroblasts (DEFs) and one-day old ADOL line 15 X 7 chickens. DNA from DEFs infected with virus at various passage levels and from buffy coat (BC) cells obtained from chickens at 3 and 8 weeks post-infection with virus at hatch was tested for the presence of REV LTR by PCR. Results from in vitro evaluation revealed the presence of REV LTR in all viruses tested even in those used at levels higher than the 40th passage. In contrast, REV LTR was not detected in BC cells obtained from infected chickens, regardless of age at testing although the incidence of MD in these chickens that were inoculated with rMd5 BAC containing REV-LTR was less than that in chickens inoculated with rMd5 BAC without LTR or with wild type Md5 strain of MDV. Results from this study suggest that REV LTR was stable in cell culture, but not in chickens; and that the loss of REV LTR following replication of virus in chickens appears to occur within the first three weeks following infection with virus at hatch. The data also suggest that the influence of REV LTR on pathogenicity of MDV may not be dependent on retention of LTR for 3 weeks or longer after infection with virus at hatch. The information is essential for studies aimed at examining the role of insertion of genes from avian retroviruses such as REV into the genome of large DNA viruses such as MDV.

4. Immunological basis for resistance and susceptibility to Marek’s disease. Mechanism of resistant to Marek's disease (MD) is not known. To decipher the immunological basis for resistance or susceptibility to MD, comparative studies between a highly susceptible (7-2) and resistant (6-3) chicken lines were conducted. Our investigation has revealed major difference in gene expression profiling between the two lines when infected with MD virus (MDV). Differential cytokine, chemokine, and other immune-related genes expression between the splenocytes of the infected chicken lines, points to the existence of a possible immunological basis for differential responses to MDV infection. In addition, immunohistochemical analysis of tissue samples collected at different time points post inoculation, revealed a major difference in CD4+ T cell population between the two chicken lines. Data indicates that there are more CD4+ T cells in line 7-2 than 6-3. This could be a possible explanation for severe susceptibility and considerable tumor development in line 7-2, as CD4+ T cells are target cell for MDV infection and transformation. Furthermore, our data indicates the level of CD8 antigen expression in CD8+ T cells was severely down regulated in line 7-2 in comparison to line 6-3. This observation is critical in understanding the essential role of cytolytic T cells in immune responses against viral infection. Differential expression of immune-related gene provides insight into possible modulation of the immune system toward an effective T cell mediated immune response against MDV infection using cytokine and chemokines as genetic adjuvant.

5. Meq is an immunosuppressive oncogene. The direct involvement of Marek's disease virus (MDV) meq oncoprotein in chicken CD4+ T cells transformation is well established. The molecular mechanism behind the transformation capacity of this virally encoded oncogene is, however, poorly understood. To provide further insight into mechanism of transformation and biological pathways involved, we conducted a comparative investigation between rMd5 and rMd5deltameq (meq deleted mutant) infected chickens. Our studies have revealed that meq expression in rMd5 (intact virus) functions as an immunosuppressive gene that results in down-regulation of many cytokines and chemokines that are essential for orchestrating an effective anti-viral immune response. In addition, the data indicates that p53, a tumor suppressive gene, is also down-regulated by rMd5 MDV. This suggests that MDV meq gene might be involved in controlling the expression levels of p53 that plays an essential role in regulating the cell cycle and tumor development. Data from our recent studies using Fowlpox virus as a delivery mechanism for meq gene expression in chicken embryonic fibroblast, indicates that meq alone is not capable of suppression of immune genes. Interaction between meq and other viral and possibly host genes are required for such suppressive function. This observation is important for understanding the MDV pathogenicity and its mechanism of immune suppression.

6. Factors leading to the evolution of Marek’s disease virus (MDV). In order for newly evolved MDV strains to become established within a flock, it seems inevitable that any new strain would need to infect and replicate in chickens previously infected with resident MDV strains. Using recombinant strains that were distinguishable by pyrosequencing and immunohistochemistry, we detected both viruses in a majority of feather and tumor samples from birds when the interval between virus challenges was short; however we only detected both viruses in a small portion of birds when the challenge interval was long. Vaccination with HVT had no significant effect on the virus frequency for either virus pair or challenge time interval, suggesting these conclusions may be applicable to vaccinated chickens in the field. These studies demonstrated superinfection for the first time with two fully virulent MDV strains and suggest that short interval challenge exposure and/or weak initial exposures may be important factors leading to superinfection; a prerequisite for the establishment of a second virus strain in the population. This model system should be useful to further elucidate the important phenomenon of virus evolution.

7. Surveillance of field flock for Marek's disease virus (MDV) with unusually high pathogenicity. Blood and tumor samples were submitted by University of Pennsylvania from a flock of 36-week old commercial white layers vaccinated with HVT and Rispens experiencing higher than normal mortality attributed to Marek’s disease. Testing for purity of virus isolates by immunohistochemistry revealed a mutation similar to two isolates submitted from nearby Pennsylvania flocks in 2007. Pathotyping to determine the virulence of the 2010 isolate is ongoing. Surveillance of field strains is critical for monitoring the evolution of MD in the field, a major concern with the poultry industry. This study will determine if the mortality in the affected flock can be attributed to virus evolution and the presence of the virus mutation may be useful for understanding the epidemiology of mutated virus strains. These findings also confirm that the T65 antibody is not specific for Rispens vaccine, therefore results when using T65 should be interpreted with caution.

8. Screening for recombinant avian leukosis viruses (ALVs) in cell cultures inoculated with various subgroups of virus. New recombinant avian leukosis viruses (ALVs) have recently been isolated from affected chickens in field outbreaks as well as from contaminated commercial Marek’s disease vaccines. Such recombination poses a problem in the diagnosis and classification of ALVs. Experiments were conducted to determine best method for screening and detection of such recombinant ALVs. Chicken embryo fibroblasts (CEFs) prepared from ADOL SPF embryos were co-infected with different concentration ratios of subgroups A and J ALV. Inoculated cultures were screened for recombination among the ALV strains. Potential recombinant viruses were purified by limiting dilution and then screened for subgroup on virus restrictive cell lines. The inability to detect recombination between ALV-A and ALV-J in cultures co-infected with both viruses suggests that conditions used in the current experiments were not suitable for recombination and other factors such as multiplicity of infection, strain and dose of virus dose should be considered. Use of PCR primers specific for envelope and LTR of subgroup of ALV following propagation on restrictive CEFs should be a useful tool in identifying recombinant ALVs, if present. The information is useful and should be helpful in designing any scheme to screen for recombinant ALVs.

9. Further studies on recombinant Marek's disease (MD) virus (MDV) strain rMd5delMeq vaccine. Vaccines have been cornerstones in the control of MD in chickens. At the present, CVI988/Rispens virus is used worldwide for controlling MD in the field. The continued evolution of MDV towards greater virulence has prompted concern that the currently available vaccine will ultimately loose efficacy in controlling MD. We have developed an oncogene Meq deleted virus, rMd5delMeq, a superior vaccine for MD in both laboratory and commercial chickens in the field. To further studies on factors which may influence rMd5delMeq vaccine efficacy, we carried out experiments on host genetics, strains of challenge virus, vaccine and challenge doses, age of challenge and maternal antibody effect. Results of these experiments demonstrated that all these factors significantly influence the efficacy of protection. The rMd5delMeq vaccine induced significant protection in five genetic lines of chickens and provides early protection and lasting immunity under challenge with a very virulent plus strain of MDV (vv+686). The information obtained from these studies is of significance for designing and comparing new vaccine candidates. In addition, these studies contribute to scientific knowledge on the pathogenesis and control of Marek’s disease.

10. Generation of a highly effective Marek’s disease virus (MDV) and infectious laryngotracheitis virus (ILTV) vaccines using bacterial chromosome (BAC) technology. Currently available commercial MDV vaccines are not highly protective against all MDV field challenges. We previously reported that we deleted both copies of the MEQ gene from a MDV BAC. The deletion virus was completely attenuated. In a protection study, the deleted virus protected chickens from a challenge with a highly virulent MDV. Our deletion virus was a much better MD vaccine than the best commercially available vaccine. We now report our attempts to extend this potential Marek’s disease (MD) vaccine clone to function as an infectious laryngotracheitis (ILT) vaccine. We cloned the gB and gJ genes from ILTV and replaced the LORF10 protein coding region of the MDV BAC with the cloned ILTV genes. Both the gB and gJ genes were stable and evidenced by measurable expression levels. Thus, we will be able to generate a polyvalent vaccine that can protect against both Marek’s disease and ILT.


Review Publications
Lee, L.F., Kreager, K.S., Arango, J., Paraguassu, A., Beckman, B., Zhang, H., Fadly, A.M., Lupiani, B., Reddy, S.M. 2010. Comparative Evaluation of Vaccine Efficacy of Recombinant Marek's Disease Virus Vaccine Lacking Meq Oncogene in Commercial Chickens. Vaccine. 28(5):1294-1299.

Suchodolski, P.F., Izumiya, Y., Lupiani, B., Ajithdoss, D.K., Lee, L.F., Kung, H., Reddy, S.M. 2010. Both Homo and Heterodimers of Marek's Disease Virus Encoded Meq Protein Contribute to Transformation of Lymphocytes in Chickens. Virology. 399(2):312-321.

Pandiri, A.R., Mays, J.K., Silva, R.F., Hunt, H.D., Reed, W.M., Fadly, A.M. 2010. Subgroup J Avian Leukosis Virus Neutralizing Antibody Escape Variants Contribute to Viral Persistence in Meat-Type Chickens. Avian Diseases. 54(2):848-856.

Silva, R.F., Dunn, J.R., Cheng, H.H., Niikura, M. 2010. A MEQ-Deleted Marek's Disease Virus Cloned as a Bacterial Artificial Chromosome Is a Highly Efficacious Vaccine. Avian Diseases. 54(2):862-869.

Jackwood, M.W., Hickle, L., Kapil, S., Silva, R.F. 2008. Vaccine Development Using Recombinant DNA Technology. Animal Agriculture's Future through Biotechnology, Part 7. Council for Agricultural Science and Technology (CAST) Issue Paper. p. 1-11.

Heidari, M., Sarson, A.J., Huebner, M., Sharif, S., Kireev, D., Zhou, H. 2010. Marek's Disease Virus-Induced Immunosuppression: Array Analysis of Chicken Immune Response Gene Expression Profiling. Viral Immunology. 23(3):309-319.

Witter, R.L., Gimeno, I.M., Pandiri, A.R., Fadly, A.M. 2010. Tumor Diagnosis Manual: The Differential Diagnosis of Lymphoid and Myeloid Tumors in the Chicken, 1st edition. Jacksonville, Florida: American Association of Avian Pathologists, Inc. p. 1-84.

Abdul-Careem, M.F., Javaheri-Vayeghan, A., Shanmuganathan, S., Haghighi, H., Read, L.R., Haq, K., Hunter, D.B., Schat, K.A., Heidari, M., Sharif, S. 2009. Establishment of an Aerosal-Based Marek's Disease Virus Infection Model. Avian Diseases. 53(3):387-391.

Abdul-Careem, M.F., Haq, K., Shanmuganthan, S., Read, L., Schat, K.A., Heidari, M., Sharif, S. 2009. Induction of Innate Host Responses in the Lungs of Chickens Following Infection With A Very Virulent Strain of Marek's Disease Virus. Virology. 393(2):250-257.

Last Modified: 4/19/2014
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